cocktail (PMA-ionomycin, eBioscience) in the presence of 1 ml/ml GolgiStop (BD) for 4 hours at 37°C.
Cells were then washed, fixed, and permeabilized
with the Cytoperm/Cytofix reagents (BD Biosciences)
according to the manufacturer’s protocol. PE-anti-
IL-21 (mhalx21, eBioscience) and its matched isotype control were then used at 1:200 to stain the
cells for 2 hours on ice. For additional ex vivo
experiments, surface-stained cells were immediately
fixed and processed for intracellular staining without PMA-ionomycin stimulation.

T-B conjugation assay

This assay was done as previously described with
minor modifications (15). Briefly, EFNB1-transduced
or control B cells were labeled with 9H-(1,3-
dichloro-9,9-dimethylacridin-2-one-7-yl (DDAO,
Invitrogen), pulsed with OVA323-339 peptide at
indicated concentrations, washed, and gently spun,
at a 2:1 ratio, together with OT-II T cells that were
previously activated in vitro for 3 to 5 days and
stained with CMF2HC (Invirogen). The cells were
incubated for 30 min at 37°C and sheer-stressed
by vortexing for 30 s before remaining T-B
conjugates were enumerated by flow cytometry.

EFNB1-Fc stimulation

EFNB1-Fc or control hIgG-Fc proteins at the indicated concentrations were used to coat 24-well
plates overnight. CD4 T cells were activated with
anti-CD3 or anti-CD28 for 48 hours, washed, and
then put into the coated plate at 106 cells per
well. After 4 hours of stimulation at 37°C, T cells
were collected for measuring IL-21 by quantitative RT-PCR.

Intravital imaging and analyses of
interactional and migratory dynamics

The surgical and imaging procedures for visualiz-ing GCs in the draining lymph node were essen-tially the same as previously described (27). Toexamine GC TFH recruitment and retention, 8 × 105Cd19+/creEfnb1fl/y or Cd19+/creEfnb1+/y CFP-expressing MD4 B cells were cotransferred with105 GFP-expressing OT-II cells to B6 recipientsthat were subsequently immunized with HEL-OVA. For virally transduced OT-II T cells, 106 cellswere cotransferred together with 5 × 105 MD4B cells to each B6 recipient. T cells were tracedsemi-automatically by Imaris (Bitplane) spot-tracking with manual correction, and the GCvolume was retrieved by creating a surface objectbased on MD4 CFP fluorescence signals. T cellmigratory tracks were then classified accordingto how they interacted with a 10-mm-wide inter-face zone, virtually defined using the Imaris cellfunction. Specifically, T cells reaching the GC-follicle border were classified according to theirpositions at the end of the 60-min observationperiod as inside the GC, at the interface, or outsideof the GC. Therefore, there were two types of GCsand three categories of cell behaviors. A 2 × 3contingency table was set up accordingly to capturethe two conditions, EFNB1-deficient or -sufficientGCs, and three categories of positioning outcomesto conduct c2 test in Prism. To measure individualTFH-GC B cell contacts, 105 dsRed-expressing OT-IIT cells were cotransferred with 7 × 105 nonfluo-rescent MD4 B cells, 5 × 104 GFP-expressing MD4B cells, and 5 × 104 CFP-expressing Cd19+/creEfnb1fl/yor Cd19+/creEfnb1+/y MD4 B cells to B6 recipientsthat were then immunized with HEL-OVA. Asintroduced in a previous study (27), the surfaceengagement index of individual TFH cells, definedas the maximal fraction of the T cell perimeterbeing engaged by a GC B cell during one con-tinued T-B contact, was used to measure theextent of T-B interactions in GCs. All imagingstudies were conducted at day 7 after immuni-zation, with a typical excitation wavelength of880 to 920 nm, an xyz dimension of 512 × 512 ×63 mm, a time resolution of 30 s per frame, and azoom factor of 1. Adobe Photoshop and AfterEffectwere used to prepare image sequences and videos.

Statistics

Statistics and graphing were done in Prism
(Graphpad). Unless indicated otherwise, two-tailed
unpaired Student’s t test was used to compare endpoint means of different groups.